The present invention relates to a method and device for quantifying the activity of an enzyme using a chromatographic test strip. More particularly, the present invention quantitates enzyme activity by transforming a substantially depletable amount of a chromogenic substrate from soluble chromogen to insoluble chromophore as the mobile phase advances, whereupon the insoluble chromophore becomes immobilized on the test strip to produce a column of color. The length and/or the intensity of the column of color are related to the activity of the enzyme. The present invention is useful for the direct determination of enzyme analytes and for the indirect determination of analytes which can be coupled to enzymes.
The prior art contains numerous examples of chromatographic methods for quantitating an analyte. See, for example, U.S. Pat. Nos. 4,298,688 (Kallies); 4,059,407 (Hochstrasser); 4,168,146 (Grubb); and 4,435,504 (Zuk). All of these examples employ soluble chromogens which are converted to soluble chromophores.
Soluble chromophores present numerous problems to the development of a quantitative chromatographic assay; namely they advance as a narrow line with the migrating solvent front, making quantitation impossible. To avoid this problem and produce a quantitative chromatographic assay, chromatographic assays were produced wherein the chromophore was developed only after migration had occurred. Although these assays avoid the migration problem, they require multi-step procedures.
Another problem with soluble chromophores is that they tend to diffuse over time. Hence, the measurement of any color must be precisely timed, particularly if the distance traveled by the color is to be proportional to the quantity of analyte present. In addition, conventional reaction kinetics dictate that results be read at a precise time during development.
Copending and co-owned application Ser. No. 118,148 filed Nov. 6, 1987 discloses a method of quantitating enzyme activity employing a chromatographic strip device. A sample containing enzyme is immobilized at a reaction site on the strip and a solution of substrate/cofactors is transported over the site to form a reaction product which is transported to a detection region beyond. The rate of reactant consumption or product formation is then determined.
Others have attempted to solve soluble chromophore problems by employing dipstick tests which do not require a mobile aqueous phase. These devices have a plurality of reagents contained therein in distinct zones (i.e. layers). Application of the sample to the test device causes the reagents to dissolve and mix uniformly over the dipstick. Color is uniformly distributed over the surface of the test device. Quantitation, if possible at all, can be done only by comparison against a standard or by instrumentation capable of reflectance or transmission spectrophotometry.